classical genetic mapping haploid mapping cross · wild type mutant wild type mutant 3 recombinants...

1

Genetic Mapping of Mutations

1. Identifying candidate genes2. Initiating positional cloning projects

Genetic Maps Have Two Properties:

• Distance between markers• Marker order

Genetic distance is determined by recombination frequencyResolution of map is determined by number of meioses scored

Classical Genetic Mapping Haploid Mapping Cross

Mutants will tend to have the a allele, B and b will be 1:1WT will tend to have the A allele, B and b will be 1:1

Markers used in initial mapping:• SSLP Markers (Simple sequence length polymorphisms)

Also called:CA-repeats, SSRs (simple sequence repeats), microsatellitesLength of CA tract differs in different strains

CACACACACACACACA

Forward

Forward

Reverse

Reverse

200 bp

206 bp

CACACACACA

• Co-dominant: both alleles can be detected in a heterozygote

• Informative in most crosses: 50-90% polymorphic among strains

• Robust markers: easily scored, reproducible banding patterns, easy transfer information between crosses and labs

Single nucleotide polymorphisms

• snip SNP’s (restriction enzyme polymorphisms)

A

G

200 bp

60 bp, 140bp

Forward

ForwardReverse

Reverse

• Co-dominant: both alleles can be detected in a heterozygote

• Abundant polymorphisms: ~1/100 bp

• Sequence information needed for assay design

• With the advent of deep sequencing, millions of SNPs can bescored in a single experiment

2

SSLP markers scored onhaploid individuals

Wild type Mutant

Wild type Mutant3 recombinants among 20 meioses = 15 cM

0 recombinants among 20 meioses = 0 cM

8 different SSLP markers scored onpools of haploid WT and mutants

Three classes of markers:• non-polymorphic (uninformative)

• polymorphic unlinked• polymorphic linked

For gel pictures from a previous year, see

http://people.fas.harvard.edu/~ianwoods/woods_hole_2010/

Testing pools with 48 primersarranged in a 96-well format

454137332925211713951

4541373329252117139514642383430262218141062

4642383430262218141062

4743393531272319151173

4743393531272319151173

4844403632282420161284

4844403632282420161284

mutant

mutant

mutant

mutant

WT

WT

WT

WT

Where did those primers come from?

Original link, now not maintained:http://zebrafish.mgh.harvard.edu/mapping/ssr_map_index.html

All data on SSLPs, available at zfin.org

~ 240 primer pairs = 5 96-well plates

http://people.fas.harvard.edu/~ianwoods/woods_hole_2010/

Original site: MGH zebrafish server

Primers used in our lab

Woods Hole Zebrafish Course 2007

3

Mapping a mutation1. Establish a polymorphic mapping cross

2. Prepare genomic DNA from wild-type and mutantsiblings

3. Analyze SSLPs on WT and mutant pools(3a optional: retest some markers on the pools)

4. Analyze putatively linked SSLPs on moderate number ofindividuals (50-100)

5. Analyze more individuals for high-resolution mapping

Diploid mapping crossX

X+/- +/-

+/- +/+

Outcross ID’d carrierto a divergent strain

Identify carriers andintercross

+/+

+/-

-/-

+/+

+/++/+

+/+

+/++/+

+/+

+/++/+

+/+

+/++/+

+/+

+/-

+/-

+/-

+/-+/-

+/-

+/-

+/-

+/-

+/-

+/-+/-

+/-

+/-

+/-

+/-

+/-

+/-+/-

+/-

+/-

+/-

+/-

+/-

+/-

+/-+/-+/-

-/-

-/-

-/-

-/-

-/-

-/- -/-

-/-

-/--/-

-/--/-

-/-

Collect embryos

Bulk Segregant Analysis on diploids-/-

-/- -/--/-

-/--/- -/--/- -/-

-/-

+/++/++/+ +/-

+/-+/-

+/-+/-

+/- +/-(Carefully) sort embryos

Prepare gDNA from embryos

Pool DNA samples (20 each)

PCR with arrayed primers

WTmut

WTmutWTmutWTmut

WT

mut

Expected results from BSA on diploidsMarker Q:Polymorphicunlinked

Marker X:Not polymorphicunlinked

Marker Y:Polymorphiclinked

Marker Z:Not polymorphiclinked

mut WT mut WT mut WT mut WT

m + m +

x

Q q Q q

m + m +

x

xx xx

m + m +x

Y y Y y

m + m +x

z z z z

Mapping a mutation1. Establish a polymorphic mapping cross

2. Prepare genomic DNA from wild-type and mutantsiblings

3. Analyze SSLPs on WT and mutant pools(3a optional: retest some markers on the pools)

4. Analyze putatively linked SSLPs on moderate number ofindividuals (50-100)

5. Analyze more individuals for high-resolution mapping

Mapping of mutant X• Incompletely characterized somite phenotype, ventrally curved body axis

• Diploid mapping cross

• PCR: 48 markers on linkage groups 4-7WT pool, mut pool

• Load and run gel (96 samples)

• Analyze BSA gels for linkage

• Analyze some markers on individuals

• Discuss follow-up experiments

4

Group 1 Group 2

Group 3 Group 4

Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

What’s next?

Test promising markers on panels of individual mutantsand wild-types

Not all markers in the following slides are “promising,” butwere chosen to show different kinds of possible results

First, the ones that were wrong . . .

5

Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z22467

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS

WILD-TYPEINDIVIDUALS

Not polymorphicUninformative marker

Test others in region, perhaps run gel longer

Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z11988

Polymorphic, not linkedLikely not located near this marker

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z8693

Polymorphic & linked?Marker segregating as an intercross

No recombinants in 16 meiosesRun more mutant individuals:

Confirm position and distance from mutation

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


6

Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z11119




1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z3057


Potential recombinants in mutant #3 = 1 out of 16 meiosesRun more mutant individuals to confirm

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z4999



1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


7

Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z7109



1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


Sample gel

Marker

Wild

-type

po

ol

Mu

tan

t po

ol

Z13936


Potential recombinants in mutant #4, 5, 7, 8 = 4 out of 16 meiosesRun more mutant individuals to confirm

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


Linked markers:Marker LG Data

Z13936 7 4 recs: #4, 5, 7, 8 (4/16)Z8693 7 0 recs (0/16)Z11119 7 0 recs (0/16)Z3057 7 1 rec: #3 (1/16)Z4999 7 1 rec: #3 (1/16)Z7109 7 1 rec: #3 (1/16)

Model of mutation location

LG 7

Z3057Z4999Z7109

Z11119Z8693 Z13936

mutation

8

1 2 3 4 5 6 7 8

- - - - - - - -

Mutant Individuals

Mutant locus

Z11119




1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


1 2 3 4 5 6 7 8

- - - - - - - -m m m m m m m m

Mutant Individuals

Z11119

Mutant locus

Z3057



1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


1 2 3 4 5 6 7 8

- - - - - - - -m m m m m m m mm m M m m m m m

Mutant Individuals

Z11119

Z3057

Mutant locus

Z13936


Potential recombinants in mutant #4, 5, 7, 8 = 4 out of 16 meiosesRun more mutant individuals to confirm

1 2 3 4 5 6 7 8

MUTANTINDIVIDUALS


9

1 2 3 4 5 6 7 8


m m m M M m M M

Mutant Individuals

Z11119

Z3057

Z13936Mutant locus

1 2 3 4 5 6 7 8


m m m M M m M M

Mutant Individuals

Z11119

Z3057

Z13936Mutant locus

X

X X X X

1 2 3 4 5 6 7 8

- - - - - - - -m m m m m m m mm m M m m m m mm m m M M m M M

Mutant Individuals

Z11119

Z3057

Z13936

Mutant locus

X

X X X XX

Overview of zebrafish genome and genomic resourcesGenome size: 1.4 x 109 bp (for comparison, C.elegans 108, Drosophila 1.7 x 108, pufferfish 0.4 x 109, mammals 3.3 x 109)

25 chromosomes

Maps and other genomic resources

pre-1994 No two genes or markers were known to be linked

1994 First genetic map (Postlethwait et al.), ~400 RAPDs, several genes and mutationsFirst mutation cloned by the candidate gene approach (Schulte-Merker et al.)

1996 Centromeres mapped, more RAPDs added, remaining gaps closed (Johnson et al.)First SSLP map (Knapik et al.), ~100 markersLarge-insert genomic libraries become available (Amemiya, Zon, Fishman et al.)Insertional mutagenesis used to clone mutated genes (Hopkins and colleagues)

1998 ~140 genes mapped (Postlethwait et al.)SSLP map expanded to ~700 markers (Knapik et al.)Large-scale generation of ESTs begins (Washington Univ. Sequencing Group)First gene identified by positional cloning (Zhang et al.)

1999 SSLP map expanded to 2000 markers (Shimoda et al.)~250 genes and ESTs genetically mapped (Gates et al.)Radiation hybrid maps become available (Geisler et al., Hukreide et al.)~200 additional genes mapped in RH panels (Geisler et al., Hukreide et al.)

2000 More than 50,000 ESTs generated (WashU)~2000 genes and ESTs genetically mapped (Woods et al.; Stanford, Oregon)~4000 genes and ESTs mapped in RH panel (WashU, Children’s, NIH, Tübingen)~50 mutations identified by candidate approach (Many groups)5-10 mutations identified by positional cloning (Many groups)Dozens of mutations cloned by insertional mutagenesis (Hopkins et al.)

2005-07 >1,400,000 ESTs defining ~20,000 genes~3400 genes and ESTs genetically mapped (Stanford, Oregon)>5000 genes mapped in RH panels (Hukreide et al. 2001, Children’s, NIH, Tübingen)Hundreds of mutated genes cloned by insertional mutagenesis (Hopkins/MIT)>200 mutated genes cloned by candidate approach and positional cloning 7,823 BAC clones sequenced, totaling 1.02 Gb of sequence (June 2007)

2010 (Almost) entire 1.4 Gb genome in high-quality assembly>11,500 genes sequenced as full-length cDNAs (NIH/ZGC)~150,000 SNPs mapped at high-resolution, used for assembly framework (Sanger)

Genome Resource Overview, continued Deep sequencing will change everything!

10

Strategy: sequence wildtype and mutant pools of genomic DNA

mut mutG G

mut +G A

+A

+A

Wild type pool Mutant pool

ATCGGCATGCATCGTAGCACGAATCGGCATGCATCGTAGCACGAATCGGCATGCATCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCATCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGA

ATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGAATCGGCATGCGTCGTAGCACGA

1!

Positional cloning: the rest of the story

http://faculty.ithaca.edu/iwoods/docs/wh!

X Today: So you have a map location … now what?

Mapped Mutant Cloned Gene

From mutant map position to cloned gene

• Refining the map location with high-resolution mapping

• Trolling for candidate genes

• Testing candidates

Refining the map position Two basic strategies: • Establish boundaries: Test other markers

- SSLPs – easy, do first - SNPs – require sequence data

• Improve resolution: Test more meioses

- generate more mutants

One advanced strategy:

• Deep sequencing of WT and mutants => SNPs = more markers to map actual mutation? esp. combined with hybrid capture . . .

2!

Data so far:

Mutant with defects in slow muscle specification

Initial Mapping:

Out of 16 meioses:

1 recombinants: Z3057, Z4999, Z7109

0 recombinants: Z8693, Z11119

4 recombinants: Z13936

What SSLPs are in the region? http://www.zfin.org!

Ciick on ‘Genetic maps’

ZFIN map query

MGH = microsatellite / SSLP map

ZFIN map view

Start close and move out both ways

3!

ZFIN marker view GenBank Marker View

Obtaining FASTA sequence Designing PCR primers

http://frodo.wi.mit.edu/primer3/!

4!

Testing for informative

SSLPs!

Informa(ve+=+polymorphic+Different+lengths+on+WT+and+mut+chromosome+

Identifying polymorphic

markers

Informa(ve+=+polymorphic+…+some+will+have+same+SSLP+allele,+

++not+good+for+mapping+

Refining the map

More fish (i.e. embryos / larvae)

= more recombinants = higher resolving power

Narrowing the critical interval

More fish = more better

5/1156

7/1156

5!

Now what?

• Identify more markers and do more high-res mapping Key point = continually refine boundaries by recombination

• Look in genome for potential candidates

What’s nearby in genome? (as a MODEL of reality)

No luck in genome sequence? (rare these days) misassembly gaps

• conserved synteny with other fish • Physical map: BAC clones • genetic or RH maps

Now what?

• Identify more markers and do more high-res mapping Key point = continually refine boundaries by recombination

• Look in genome for potential candidates

What’s nearby in genome? (as a MODEL of reality)

No luck in genome sequence? (rare these days) misassembly gaps

• conserved synteny with other fish • Physical map: BAC clones • genetic or RH maps

What’s nearby in the genome? http://www.ensembl.org/Danio_rerio/!

Ensembl marker search

No luck! Let’s try a sequence-based search: BLAST/BLAT!

6!

Ensembl BLAT search

Scroll down And hit “Run”!

Genome assembly

7!

Good candidate? External references

calca at ZFIN calca expression

motor neuron expression muscle phenotype

what if . . . signal from motor neurons to developing muscle?!

8!

What’s known about calca?

http://www.ncbi.nlm.nih.gov/gene!

What’s known about calca?

Cool new biology: it’s a secreted peptide with a novel role in directing slow muscle specification! Alert Cell, Science, and Nature!!

How to test if this is the right gene?

Is calca the right gene? High resolution mapping

- no recombinants between mutation and gene in lots of meioses

Phenocopy with MO injection or noncomplementation with another allele

Rescue with mRNA injection

Find mutation in coding sequence

Picking the right strategy often is determined by balance of . . .

- Available Resources - Number of Candidates

These are often determined by size of candidate interval

9!

Now what?

Test potential candidates

• Turn the candidate into a new map marker - could it be the right gene? - if not, can it narrow your interval?

How to turn it into a map marker?

What’s a good candidate?

Now what?

Test potential candidates

• Turn the candidate into a new map marker - could it be the right gene? - if not, can it narrow your interval?

How to turn it into a map marker?

What’s a good candidate?

Single nucleotide polymorphisms

A

G

200 bp

60 bp, 140bp

Forward

Forward Reverse

Reverse

SNPs+=+~+1+/+250+bp+in+genome+

Generating map markers from ESTs/Genes/other sequences

• Find or design primers for PCR (from gDNA)

• Sequence PCR product on WT and mut

• Find RE polymorphism

10!

Obtaining gDNA from cDNA sequence: exporting from genome!

http://genome.ucsc.edu/!




11!





12!


Beware of shotgun (non-BAC, i.e. large clone) assembly

Here there be Monsters

Safe Sailing (mostly)

Designing PCR primers

http://frodo.wi.mit.edu/primer3/!

PCR primers

Amplify from WT and mut, sequence . . .

Locating a SNP to map

. . . run on your mapping panel - still a candidate? (0 recombinants) - narrow the candidate interval?

13!

Identifying a restriction enzyme to map your SNP

http://helix.wustl.edu/dcaps/dcaps.html

dCAPS results!

Striking the right balance in positional cloning

Mapping:

lots of fish, lots of PCR, lots of gels should always give you an unambiguous answer

Functional:

Sequencing => often done concomitantly with mapping

mRNA cloning/rescue Morpholinos => time, money Ambiguous, easy to make up lots of stories

Mapping vs. Functional follow-up!

What if ZF genome turns out to be a dead end?

• Check other fish genomes

- more candidate genes? - fix a gap in the ZF data

• Check genetic/RH maps on ZFIN

• Start a chromosome walk

- iterative BAC screening

14!

What if ZF genome turns out to be a dead end?

• Check other fish genomes

Pufferfish (Tetraodon, Fugu)

- smaller, more compact genome - good for getting enhancer regions

Tetraodon calca region

More Candidates to test: find and map zebrafish orthologs!

Today: So you have a map location … now what? Mapped Mutant Cloned Gene

Tomorrow’s bioinformatics practical:

1) Positional cloning in 2 (mostly) easy steps

2) Morpholinos (ATG, Splice) and rescue

3) Zebrafish orthologs of your favorite human genes Identification of enhancer elements Transgenic Lines

4) Doing cool things in big batches

classical genetic mapping haploid mapping cross · wild type mutant wild type mutant 3 recombinants...

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